Cigarette smoking is an important cause of vascular and pulmonary morbidity and mortality and is increased among veterans. Both systemic and pulmonary endothelial cell (EC) injuries are important consequences of Cigarette Smoke (CS) exposure. CS is associated with an increased incidence of Acute Respiratory Distress Syndrome (ARDS), with the hallmark of increased endothelial permeability. Previous studies from our laboratory indicate that CS increases lung vascular permeability in mice and in cultured lung vascular EC; an effect involving decreased focal adhesion kinase (FAK) and RhoA GTPase activation. However, the components of CS involved in this injury are unknown. Acrolein is a highly reactive a, - unsaturated aldehyde that is present in CS and in exhaled breath condensates and blood of smokers. Our preliminary data indicate that acrolein, like CS, increases lung vascular EC permeability in vivo and in vitro; disrupts actin stress fibers, adherens junctions, and focal adhesion complexes; and decreases RhoA and FAK activation. CS- and acrolein-induced lung endothelial injury is prevented by inhibition of aldehyde stress in vivo and in vitro. Our Overall Objective is to understand the mechanism of CS-induced lung endothelial cell dysfunction. We hypothesize that acrolein mediates CS-induced EC dysfunction resulting in increased permeability pulmonary edema via carbonylation of RhoA and FAK in lung endothelial cells. Aim 1. We will determine: The effects of acrolein on lung microvascular barrier function in vivo and in vitro, including: a. The effects f acrolein on mouse lung microvascular permeability. b. The effects of acrolein on RhoA and FAK carbonylation and inactivation and the mechanism(s) of these changes. c. If inhibition of protein carbonylation attenuates CS- and acrolein-induced increased vascular permeability in vivo and in cultured lung EC. Circulating extracellular vesicles (EV) bearing EC surface markers have been isolated from plasma of smokers and our preliminary data indicate that there are increased circulating EV in plasma of mice exposed to CS. Our preliminary data show that EV isolated from EC treated with acrolein significantly increased EC permeability, compared to EV isolated from EC treated with vehicle. Therefore, we also hypothesize that acrolein in CS decreases lung endothelial barrier function via EV. Aim 2. We will determine the role of circulating and lung EV in CS- and acrolein-induced lung injury, including: a. Characterization of formation of EV in response to CS and acrolein exposure. b. The effects of circulating and lung EV from CS- or acrolein-exposed mice on lung EC barrier function in healthy mice and in cultured mouse lung microvascular EC (LMVEC). c. The effect of EV isolated from CS- or acrolein-exposed LMVEC on carbonylation and activities of RhoA and FAK in unexposed LMVEC and in purified proteins. d. If inhibition of protein carbonylation prevents lung vascular injury caused by EV from CS- or acrolein-exposed mice. These studies will comprehensively assess the effects of acrolein on lung EC and EV and their roles in CS-induced lung vascular permeability. Better understanding of CS-induced lung EC injury is likely to enhance understanding of both ARDS and more chronic CS-induced diseases that may involve endothelial injury, such as COPD.